PROJECT SUMMARY The question of how the fetus and placenta avoid rejection by the maternal immune system has puzzled generations of immunologists and reproductive biologists. A great deal of work on this immunological paradox has focused on the problem of how the maternal immune system tolerates fetal alloantigens. However, another problem during pregnancy entails the exposure of the mother to numerous newly-induced or strongly upregulated self-antigens encoded by the mother?s own genome. Many of these ?pregnancy associated antigens? (PAAs) have not been produced since the mother herself was a fetus with a placenta and are only encountered in postnatal life when she becomes pregnant. A crucial component in imposing tolerance to self- antigens is the autoimmune regulator gene (Aire), which promotes the expression of tissue-restricted antigens in medullary thymic epithelial cells (mTECs) and extrathymic cells Aire expressing cells (eTACs), leading to clonal deletion or Treg conversion of self-reactive T cells. The role of Aire in supporting healthy pregnancy has not yet been explored. Surprisingly, we found that selective depletion of maternal Aire-expressing cells in mice during early in pregnancy results in a dramatic phenotype of pregnancy loss (both failure to become pregnant after plugging and early embryo resorption), along with a significant increase in the conventional T cell to Treg cell ratio in the thymus and an influx of maternal T cells into the resorbing uterus. These data are consistent with other hints in the literature such as the presence of anti-placental antibodies and pregnancy complications (such as fetoplacental insufficiency) in patients with Aire deficiency. Based on these data, we hypothesize that Aire-mediated expression of PAAs in mTECs and eTACs supports healthy pregnancy by promoting deletion of PAA-reactive T cells and induction of PAA-specific Tregs. By extension, immune responses to these antigens in the absence of Aire may play a role in pregnancy complications. This hypothesis shifts the current paradigm regarding maternal-fetal tolerance from focusing on the potential threat of alloantigens to the unknown potential value of imposing tolerance to a unique set of PAAs encoded by the maternal genome. In this proposal, we will first assess the temporal role of Aire during early vs late pregnancy and the relative contributions of mTECs vs eTACs in supporting healthy pregnancy (Aim 1). We will use a combination of unbiased biochemical and transcriptomic analyses to identify putative Aire-regulated PAAs and validate their expression in mice and in patients with infertility (Aim 2). Lastly, we will investigate the function of Aire in generating PAA-specific Tregs using a novel transgenic mouse to express a model antigen under the control of Aire in maternal mTECs and eTACs (Aim 3). Our short-term goal is to understand the mechanisms by which Aire supports healthy pregnancy. Our long-term goal is to identify specific PAAs and immune derangements in patients with infertility. Responses to these questions could provide key insights that will lead to targeted therapies for this important health problem.